Refolding Record:
| Protein | |
|---|---|
| Protein Name | Extracellular domain of myelin oligodendrocyte glycoprotein |
| Abbreviated Name | ED-MOG |
| SCOP Family | Intergral-membrane protein |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | Q61885 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 248 |
| Molecular Weight | 28296.0 |
| Pi | 8.16 |
| Molecular Weight | 28296.0 |
| Disulphides | Unknown |
| Full Sequence |
MACLWSFSWPSCFLSLLLLLLLQLSCSYAGQFRYIGPGYPIRALVGDEAELPCRISPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPEYRGRTELLKETISEGKVTLRIGNVRFSDEGGYTCFFRDHSYQEEAAMELKVEDPFYWVNPGVLTLIALVPTILLQVSVGLVFLFLGHRLRGKLRAEVENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Linares, David (2004) Protein Expression and Purification, 34, 249-256 |
| Project Aim | Protein refolding |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein expressed and purified in native conformation prior to denaturation and refolding. |
| Expression Host | None |
| Expression Strain | None |
| Expression Temp | 0.0 |
| Expression Time | 0 |
| Expression Vector | |
| Expression Protocol | |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | 8M Urea; 100mM NaH2PO4; 10mM Tris-HCl, pH 6.3 |
| Solubilization Buffer | 8M Urea; 100mM NaH2PO4; 10mM Tris-HCl, pH8.0 |
| Refolding Buffer | 8M Urea; 100mM NaH2PO4; 10mM Tris-HCl, 0-100% gradient |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | GSSG/beta mercaptoethanol |
| Redox Agent Concentration | 2mM glutathione,2mM glutathione |
| Refolding Protocol | Refolding step was carried out in the Ni-NTA column before elution,using overnight linear gradient of 0-100%.Use solubilisation buffer with 13mM beta-mercaptoethanol, finishing with refolding buffer containing 2mM reduced glutathione and 0.2mM oxidised glutathione. Remove thiol pair with additional gradient of 200ml refolding buffer for 3h. Protein eluted from Ni-NTA column using 100mM Na2H2PO4, 10mM Tris-HCl, pH 8.0, 300mM imidazole.Protein is pooled and dialysed against sodium phosphate for 2d, 4 degrees celcius. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |